The introduction of luminescent semiconductor nanocrystals or quantum dots (QDs) to biology has provided researchers with novel fluorescent tools for potentially achieving advances in imaging, sensing, and for developing optical barcodes. See, for example, U.S. Patent Application Publication No. 2008/0087843, which is incorporated herein by reference. This arises from the unique photophysical properties that these fluorophores provide including: size-tunable narrow, symmetrical photoluminescence (PL, full-width at half-maximum ˜25-40 nm) and broad absorption spectra that increase towards shorter wavelengths. Using different semiconductor combinations it is possible to prepare nanocrystals with emissions ranging from the UV to well into the near infrared region of the optical spectrum. QDs also exhibit high quantum yields, a pronounced resistance to chemical degradation, and high photo-bleaching thresholds. A particularly useful property is that multiple QDs present in the same sample can be efficiently excited at a single wavelength far removed (>100 nm) from their respective emissions. This makes QDs directly amenable to signal multiplexing, i.e., the simultaneous detection of multiple concurrent fluorescent emissions or channels.
Simultaneous detection of multiple independent fluorescent signals or signal multiplexing has the potential to significantly improve bioassay throughput and to allow visualization of concurrent cellular events. Applications based on signal multiplexing, however, have been previously been difficult to achieve in practice due to challenges in both implementing hardware and the photophysical liabilities associated with available organic dye and protein fluorophores.